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This protocol describes how to inoculate C57BL/6J mice with the EGD strain of Listeria monocytogenes (L. monocytogenes) and to measure interferon-γ (IFN-γ) responses by natural killer (NK) cells, natural killer T (NKT) cells, and adaptive T lymphocytes post-infection. This protocol also describes how to conduct survival studies in mice after infection with a modified LD50 dose of the pathogen.
L. monocytogenes is a gram-positive bacterium that is a cause of food borne disease in humans. Experimental infection of mice with this pathogen has been highly informative on the role of innate and adaptive immune cells and specific cytokines in host immunity against intracellular pathogens. Production of IFN-γ by innate cells during sublethal infection with L. monocytogenes is important for activating macrophages and early control of the pathogen1-3. In addition, IFN-γ production by adaptive memory lymphocytes is important for priming the activation of innate cells upon reinfection4. The L. monocytogenes infection model thus serves as a great tool for investigating whether new therapies that are designed to increase IFN-γ production have an impact on IFN-γ responses in vivo and have productive biological effects such as increasing bacterial clearance or improving mouse survival from infection. Described here is a basic protocol for how to conduct intraperitoneal infections of C57BL/6J mice with the EGD strain of L. monocytogenes and to measure IFN-γ production by NK cells, NKT cells, and adaptive lymphocytes by flow cytometry. In addition, procedures are described to: (1) grow and prepare the bacteria for inoculation, (2) measure bacterial load in the spleen and liver, and (3) measure animal survival to endpoints. Representative data are also provided to illustrate how this infection model can be used to test the effect of specific agents on IFN-γ responses to L. monocytogenes and survival of mice from this infection.
IFN-γ is a cytokine that is crucial for mediating immunity against intracellular pathogens and for controlling tumor growth5. The importance of this cytokine in bacterial resistance is evident in the observation that humans with mutations in the IFN-γ signaling pathway are highly susceptible to infection with mycobacteria and salmonellae6. Similarly, mice deficient in either IFN-γ or the IFN-γ receptor exhibit defects in resistance to mycobacteria7-9 and other intracellular pathogens including L. monocytogenes10,11, Leishmania major12, Salmonella typhimurium13
Safety Statement
This protocol describes infection of mice with live L. monocytogenes. The pathogen is handled safely under BSL2 conditions by trained personnel who are not immunocompromised. Immunocompromised people include pregnant women, the elderly and individuals who are HIV-infected or have chronic conditions that require treatment with immunosuppressive therapy. Personnel should don a protective lab coat or gown, gloves, mask, and eye protection while handling infected samples. The work described herein was performed under BSL2 conditions under a certificate (#32876) that was issued by the University Health Network (UHN) Biosa....
Figure 3 presents some typical flow cytometry staining of IFN-γ in splenic NK and NKT cells at 24 hr post-infection with 105 CFU of the pathogen. This figure also illustrates the gating strategy for the staining panel described in Table 2. Figure 4 shows some representative data that were obtained in one experiment where male mice were treated with the PPARα antagonist IS001 or vehicle control, infected with 105<.......
Here we describe a protocol of how to carry out a basic experimental infection with the EGD strain of L. monocytogenes25 in male or female C57BL/6J mice. This protocol was set up for the purpose of studying the effect of a novel small molecule IS001 on IFN-γ production by innate and adaptive lymphocytes in vivo31. By monitoring bacterial clearance and survival post-infection, insights were gained into how these changes in IFN-γ impacted the host's ability to control t.......
No conflicts of interest to disclose.
Development of this protocol was supported by an operating grant from CIHR (MOP97807) to SED.
....Name | Company | Catalog Number | Comments |
Brain Heart Infusion Broth, Modified | BD | 299070 | any brand should be appropriate |
Agar | BD | 214010 | any brand should be appropriate |
Triton X-100 | Sigma-Aldrich | X100 | any brand should be appropriate |
1xPBS | Sigma | D8537 | any brand should be appropriate |
TissueLyser II | Qiagen | 85300 | any brand should be appropriate |
Ammonium Chloride (NH3Cl) | any brand should be appropriate | ||
KHCO3 | any brand should be appropriate | ||
Na2EDTA | any brand should be appropriate | ||
RPMI 1640 | Gibco | 22400089 | any brand should be appropriate |
Fetal Bovine Serum | Gibco | 12483 | Before use, heat-inactivate at 56 °C for 30 min |
L-glutamine | Gibco | 25030 | any brand should be appropriate |
Non-essential amino acids | Gibco | 11140 | any brand should be appropriate |
Penicillin/Streptomycin | Gibco | 15140 | any brand should be appropriate |
GolgiStop Protein Transport Inhibitor (containing Monensin) | BD | 554724 | Use 4 μl in 6 ml cell culture |
16% Paraformaledehye | Electron Microscopy Sciences | 15710 | Dilute to 4% PFA in ddH20 or 1xPBS |
10 x Perm/Wash buffer | BD | 554723 | Dilute 10x in ddH20 |
Fc block, Anti-Mouse CD16/CD32 Purified | eBioscience | 14-0161 | Dilute 1:50 |
Fixable Viability Dye eFluor 506 | eBioscience | 65-0866 | Dilute 1:1000 (we have also used viability dyes from Molecular Probes) |
anti-Mouse CD4-PE-Cy5 (GK1.5) | eBioscience | 15-0041 | Manufacturer recommends a certain test size; however this should be titrated before use. |
anti-Mouse CD8-FITC (53-6.7) | eBioscience | 11-0081 | Manufacturer recommends a certain test size; however this should be titrated before use. |
PBS57/mCD1d tetramer-APC | NIH Tetramer Core Facility | N/A | Obtained as a gift from the facility |
anti-Mouse TCRβ-PE-Cy7 (H56-597) | eBioscience | 25-5961 | Manufacturer recommends a certain test size; however this should be titrated before use. |
anti-Mouse NKp46-APC-eFluor780 (29A1.4) | eBioscience | 47-3351 | Manufacturer recommends a certain test size; however this should be titrated before use. |
anti-Mouse CD45 PE-Cyanine7 (30-F11) | eBioscience | 25-0451 | Manufacturer recommends a certain test size; however this should be titrated before use. |
anti-Mouse IFN gamma-PE (XMG1.2) | eBioscience | 12-7311 | Manufacturer recommends a certain test size; however this should be titrated before use. |
OneComp eBeads | eBioscience | 01-1111 | Manufacturer recommends a certain test size; however this should be titrated before use. |
Mouse IFN gamma ELISA kit | eBioscience | 88-7314 | Used for measuring the interferon gamma in the culture supernatant |
50 mL vented tubes for culture | Used for culturing the bacteria, any brand should be appropriate | ||
1.5 ml microcentrifuge tubes | any brand should be appropriate | ||
bacterial petri dishes | any brand should be appropriate | ||
2 ml cyrovials | any brand should be appropriate | ||
UV spectrometer | any brand should be appropriate | ||
safety engineered needles | any brand should be appropriate | ||
C57BL6/J | Jackson laboratories | Stock#000664 | Order for arrival at 7 wks |
Bleach | For decontamination | ||
70% Ethanol | For decontamination | ||
Glass beads | any brand should be appropriate | ||
Centrifuge | rotor, buckets, bucket covers. | ||
Microcentrifuge | any brand should be appropriate | ||
Sterile Glycerol | any brand should be appropriate | ||
Pipette Tips | any brand should be appropriate | ||
Pipette | any brand should be appropriate | ||
Surgical instruments | any brand should be appropriate | ||
70 micron strainers | any brand should be appropriate | ||
3 ml syringe | any brand should be appropriate | ||
Pipette gun | any brand should be appropriate | ||
Filtration Units | any brand should be appropriate | ||
Trypan Blue | Dilute 1 to 9 in ddH20, any brand should be appropriate | ||
Hemocytometer | any brand should be appropriate | ||
Round bottomed plates | any brand should be appropriate | ||
FACs tubes | BD | ||
BD LSR II | BD | Any flow cytometer could be used for acquisition that has an appropriate laser configuration and filter set to discriminate the fluorochormes | |
Flowjo software | Treestar | Used for data analysis. Other types of data analysis software will also be appropriate | |
Multichannel pipettor (0-300 µl) | Eppendorf | Used for washing cells and adding antibodies during flow cytometry staining | |
Acetic Acid | Used for washing glass beads, any brand should be appropriate | ||
Microbank Bacterial Preservation System | Pro-lab Diagnositics | Used as an alternative to glycerol stocks for long-term storage of bacteria |
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